Monoclonal antibodies selectively bind tumor cell differentiating antigens in vitro and in vivo. Natural effector mechanisms often do not mediate killing of monoclonal antibody bound cells so we have devised methods of linking extremely toxic proteins to the antibodies to selectively kill tumor cells. Two methods of coupling toxic proteins, like ricin to antibodies, have been used to kill antigen positive cells in vitro. Ricin has two subunits, the A subunit blocks protein synthesis when in the cytosol and the B subunit binds galactose groups on all cell surfaces but also facilitates the transport of ricin A chain to the cytosol. 1) Linkage of the ricin A chain to antibodies yields reagents with low nontarget cell toxicity but target cell toxicity too slow for in vivo applications; 2) Linkage of intact ricin to antibodies results in very potent target cell toxicity but the nontarget cell killing must be prevented by a ligand which blocks ricin B chain binding to cells. This has limited to application to in vitro situations where 100 mM lactose can block ricin binding. We are testing several new approaches to apply immunotoxins in vivo. 1) Cloning of toxins then altering their structure at the gene level to decrease nontarget cell toxicity; 2) Chemical modification of ricin to determine the location of the ricin galactose binding site and to possibly improve efficacy of ricin linked to antibodies; 3) Develop new ways to block the nontarget cell toxicity of ricin in vivo. We have discovered a monoclonal antibody which blocks the ricin galactose binding site similar to lactose. Preliminary in vivo trials in guinea pigs show over a 2 fold extension of survival time with intact ricin immunotoxins with no toxicity to animals. Anticancer trials of these new approaches are ongoing.